Humanized anti clever-1 antibodies and their use

ABSTRACT

This invention relates to an agent and a humanized antibody or single chain Fv or Fab fragment capable of binding to human CLEVER-1 recognizing an epitope of CLEVER-1, wherein the epitope is discontinuous and comprises the sequences: PFTVLVPSVSSFSSR and QEITVTFNQFTK. This invention relates also an agent capable of binding to an epitope of human CLEVER-1 for use in removing tumour or antigen induced immunosuppression. Further, the invention relates to a pharmaceutical composition comprising the agent capable of binding to human CLEVER-1 and an appropriate excipient.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a national stage filing under 35 U.S.C. § 371of PCT/FI2017/050285, filed 18 Apr. 2017, which in turn claims priorityto Finnish Patent Application Nos. 20165335, filed 18 Apr. 2016, and20165336, filed 18 Apr. 2016, each of which are incorporated herein byreference.

FIELD OF THE INVENTION

This invention relates to agents specific for CLEVER-1 protein byrecognizing a specific CLEVER-1 epitope and uses thereof.

BACKGROUND OF THE INVENTION

The publications and other materials used herein to illuminate thebackground of the invention, and in particular, cases to provideadditional details respecting the practice, are incorporated byreference.

CLEVER-1 is a protein disclosed in WO 03/057130, Common LymphaticEndothelial and Vascular Endothelial Receptor-1. It is a binding proteinthat mediates adhesion of lymphocytes to endothelium in both thesystemic vasculature and in the lymphatics. By blocking the interactionof CLEVER-1 and its lymphocyte substrate it is possible tosimultaneously control lymphocyte recirculation and lymphocytemigration, and related conditions such as inflammation, at the site oflymphocyte influx into, and efflux from, the tissues.

WO 03/057130 further discloses that CLEVER-1 mediates binding of othertypes of leukocytes such as monocytes and granulocytes to HEV-likevessels. Thus, by blocking the interaction of CLEVER-1 and malignanttumour cells it became possible to control metastasis by preventingmalignant cells that bind to CLEVER-1 from being taken up by thelymphatic vessels, and thus to prevent spread of the malignancy into thelymph nodes.

CLEVER-1, i.e. Stabilin-1, has been reviewed by Kzhyshkowska J. (2010),TheScientificWorldJOURNAL 10, 2039-2053. Suppression of Th1 Lymphocytesby CLEVER-1 has been recently disclosed by Palani et al. (2016), Journalof Immunology 196: 115-123.

WO 2010/122217 discloses a subtype of macrophages in tumours, in theplacenta, and in the blood of pregnant women. The subtype of macrophagesis defined as a CLEVER-1 positive macrophage and proposed as type 3macrophage. By modulating, i.e. counteracting or stimulating,respectively, the CLEVER-1 receptor in this cell the immune system in anindividual can be affected. Counteracting or down-regulation of thereceptor reduces the size of malignant tumour and/or malignant tumourgrowth. Stimulating or upregulating the receptor is useful in generationof fetomaternal tolerance and for prevention of pregnancy complications.

The mechanisms of tumour-associated macrophages (TAMs) is also disclosedin the publication by Noy R. and Pollard J. W., “Tumour-AssociatedMacrophages: From Mechanisms to Therapy”, published in Immunity 41, Jul.17, 2014, p. 49-61. M2 macrophages predominate in human cancers andstimulate tumour growth, but these tumour promoting macrophages can bemodulated into tumour growth-inhibiting macrophages, called also as M1macrophages or pro-inflammatory macrophages, aiming to slow or stopcancer growth. However, it has been noticed that the attempts to treatcancers with the currently available therapeutics aiming at targetingTAMs were accompanied by undesired side effects, e.g. the macrophagetherapeutic approaches may have systemic toxicities or paradoxicallypromote tumour growth, as they target all macrophages.

Particularly preferred CLEVER-1 antagonist monoclonal antibodies 3-266(DSM ACC2519) and 3-372 (DSM ACC2520), both deposited under the terms ofthe Budapest Treaty on the International Recognition of the Deposit ofMicro-organisms for the Purposes of Patent Procedure on Aug. 21, 2001,with DSMZ-Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH,Mascheroder Weg 1 b, D-38124 Braunschweig, are disclosed in WO03/057130.

OBJECT AND SUMMARY OF THE INVENTION

One object of the present invention is to provide an agent capable ofbinding to a specific epitope of human CLEVER-1. Especially, it has beenfound out that an agent capable of binding to a specific epitope ofhuman CLEVER-1 can be used to activate macrophages to switch theirphenotype from M2 macrophages into M1 macrophages.

Further, an object of the invention is to provide a humanized antibodyor humanized single chain Fv or Fab fragment for binding to humanCLEVER-1 with an increased binding activity in comparison of monoclonalantibody 3-372 (DSM ACC2520 deposited at DSMZ-Deutsche Sammlung vonMikroorganismen and Zellkulturen GmbH on Aug. 21, 2001).

Therefore, the present invention provides an agent capable of binding toan epitope of human CLEVER-1, wherein the epitope is discontinuous andcomprises the sequences:

(SEQ ID NO: 1) PFTVLVPSVSSFSSR, and (SEQ ID NO: 2) QEITVTFNQFTK.

Especially, the present invention provides an agent capable of bindingto human CLEVER-1 recognizing an epitope of CLEVER-1, wherein theepitope is discontinuous and comprises the sequences:

(SEQ ID NO: 1) PFTVLVPSVSSFSSR, and (SEQ ID NO: 2) QEITVTFNQFTK,

and the agent comprises sequences of complementarity determining regions(CDRs) binding to said epitope sequences selected from the groupconsisting of

(SEQ ID NO: 7) TSGMGIG, (SEQ ID NO: 8) HIWWDDDKRYNPALKS, (SEQ ID NO: 9)HYGYDPYYAMDY, (SEQ ID NO: 10) TASSSVSSSYLH, (SEQ ID NO: 11) RTSNLAS, and(SEQ ID NO: 12) HQYHRSPPT.

According to the invention, an agent capable of binding to humanCLEVER-1 recognizing an epitope of CLEVER-1 defined in the presentapplication may be selected from the group consisting of an antibody,single chain Fv or Fab fragment(s), peptide(s) or any othermacromolecule having an adequate affinity to bind to said epitope.

In one aspect the present invention provides an agent capable of bindingto human CLEVER-1 in an individual for use in removing tumour or antigeninduced immunosuppression by modulating M2 macrophages into M1macrophages, wherein the agent binds to an epitope of human CLEVER-1,which epitope is discontinuous and comprises the sequences:

(SEQ ID NO: 1) PFTVLVPSVSSFSSR, and (SEQ ID NO: 2) QEITVTFNQFTK.

An agent according to the invention capable of binding to human CLEVER-1on TAMs, preferably to specific epitope sequences on CLEVER-1, issuitable for use in treating or preventing cancer by reducing size ofmalignant tumour; by reducing malignant tumour growth in an individual;and/or by inhibiting cancer cell transmigration and metastasisformation, wherein immune suppression around the malignant growth isremoved by modulating M2 macrophages into M1 macrophages.

An agent according to the invention capable of binding to humanCLEVER-1, preferably to specific epitope sequences on CLEVER-1, is alsosuitable for use in treating chronic infections in an individual,wherein immune suppression against the infective antigens is removed bymodulating M2 macrophages into M1 macrophages.

An agent according to the invention capable of binding to humanCLEVER-1, preferably to specific epitope sequences on CLEVER-1, is alsosuitable for use as an adjuvant of a vaccine, wherein immune suppressionagainst vaccine antigens is removed by modulating M2 macrophages into M1macrophages.

In another aspect, the invention provides a humanized antibody or singlechain Fv or Fab fragment capable of binding to an epitope of humanCLEVER-1, wherein the epitope is discontinuous and comprises thesequences:

(SEQ ID NO: 1) PFTVLVPSVSSFSSR, and (SEQ ID NO: 2) QEITVTFNQFTK,

and said antibody or single chain Fv or Fab fragment comprises

a) constant regions of human IgG4 heavy chain and kappa light chain, and

b) one or more of the following sequences of complementarity determiningregions (CDRs)

i) of the heavy chain CDR 1: (SEQ ID NO: 7) TSGMGIG, and/or CDR 2:(SEQ ID NO: 8) HIWWDDDKRYNPALKS, and/or CDR 3: (SEQ ID NO: 9)HYGYDPYYAMDY; and ii) of the light chain CDR 1: (SEQ ID NO: 10)TASSSVSSSYLH, and/or CDR 2: (SEQ ID NO: 11) RTSNLAS, and/or CDR 3:(SEQ ID NO: 12) HQYHRSPPT.

Another object of the present invention is also to provide apharmaceutical composition comprising the agent capable of binding tohuman CLEVER-1 or the humanized antibody or the single chain F_(V) orFab fragment according to the invention and an appropriate excipient.

The present invention also provides a pharmaceutical compositioncomprising the agent capable of binding to human CLEVER-1 or thehumanized antibody or the single chain F_(V) or Fab fragment as definedabove and an appropriate excipient for use in removing tumour or antigeninduced immunosuppression.

A pharmaceutical composition according to the invention is suitable foruse in treating or preventing cancer by reducing size of malignanttumour; by reducing malignant tumour growth in an individual; and/or byinhibiting cancer cell transmigration and metastasis formation. Apharmaceutical composition according to the invention is also suitablefor use treatment of chronic infections in an individual or for use asan adjuvant of a vaccine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a and 1b illustrate heatmap representation of results obtainedfor antibody 3-266 and antibody AK FUMM 9-11.

FIG. 2 illustrates heatmap representation of results obtained forantibody FU-HI-3-372.

FIG. 3 illustrates schematically the domain organization of CLEVER-1positions of identified binding motifs.

FIG. 4 illustrates 1% agarose gel separation of hybridoma 3-372 RT-PCRproducts.

FIG. 5 illustrated Coomassie Blue-stained SDS-PAGE gel of proteinA-purified chimeric 3-372 IgG4.

FIG. 6 illustrates CLEVER-1 competition ELISA.

FIG. 7 illustrates Antitope pANT vector diagram.

FIG. 8 illustrates Coomassie Blue-stained SDS-PAGE gel of selectedprotein A-purified antibodies.

FIG. 9 illustrates CLEVER-1 competition ELISA.

FIG. 10A shows results of the determination of HLA-DR expression fromCD14 positive cells and FIG. 10B shows results of soluble TNF-alphameasured from the culture medium using a TNF-alpha ELISA kit(Invitrogen).

FIG. 11A shows TAM re-polarization in syngeneic E0771 mammary carcinomasafter administration of an antibody binding to CLEVER-1 and FIG. 11Bshows increased secretion of TNF-alpha on TAMs from E0771 syngeneicmammary carcinoma after administration of an antibody binding toCLEVER-1.

FIG. 12 illustrates that CLEVER-1 ligation with 9-11 and 3-372antibodies promotes opposing effects on mTOR and c-Jun signalling inhuman peripheral blood monocytes.

DETAILED DESCRIPTION OF THE INVENTION

Terms

The term “an agent capable of binding to an epitope of human CLEVER-1”refers to agents including antibodies and fragments thereof, peptides orthe like, which are capable of binding to specific epitope sequencesdefined in the present application. The agent may also be any othermacromolecule having an adequate affinity to bind to said epitope.

The term “an antibody or a fragment thereof” is used in the broadestsense to cover an antibody or a fragment thereof which are capable tobind CLEVER-1 molecule in an individual. Especially, it shall beunderstood to include chimeric, humanized or primatized antibodies, aswell as antibody fragments and single chain antibodies (e.g. Fab, Fv),so long they exhibit the desired biological activities.

The term humanized antibody refers to any antibody wherein the constantregions of non-human antibodies have been fully substituted with thehuman form of the constant regions, and at least parts of the variableregions of the non-human antibodies, excluding the three loops of aminoacid sequences at the outside of each variable region that bind to thetarget structure, have been fully or partially substituted withcorresponding parts of human antibodies. Thus, in particular, anyantibody named by the naming scheme for the World Health Organization'sInternational Nonproprietary Names (INN) or the United States AdoptedNames (USAN) for pharmaceuticals with substems -xizu- or -zu- is in thisapplication referred to as a humanized antibody.

The term variable domain, also referred to as the F_(V) region, is themost important region for binding to antigens. To be specific, variableloops of β-strands, three on each light (V_(L)) and heavy (V_(H)) chain,are responsible for binding to the antigen. These loops are referred toas the complementarity determining regions (CDRs).

The term single-chain Fv fragment or scFv refers to fragments that areobtained by connecting the V_(H) and the V_(L) domains by a linker in asingle polypeptide. The term humanized single-chain Fv fragment or scFvrefers, in analogy with the definition of the term humanized antibodyabove, to any single-chain Fv fragment or scFv wherein the constantregions originating from non-human antibodies have been fullysubstituted with the human form of the constant regions, and at leastparts of the variable regions originating from non-human antibodies,excluding the three loops of amino acid sequences at the outside of eachvariable region that bind to the target structure, have been fully orpartially substituted with corresponding parts of human antibodies.

The term Fab fragment refers to a region on an antibody that binds toantigens. The term humanized Fab fragment refers, also in analogy withthe definition of the term humanized antibody above, to any Fab fragmentwherein the constant regions originating from non-human antibodies havebeen fully substituted with the human form of the constant regions, andat least parts of the variable regions originating from non-humanantibodies, excluding the three loops of amino acid sequences at theoutside of each variable region that bind to the target structure, havebeen fully or partially substituted with corresponding parts of humanantibodies.

The term “peptide” refers to any peptide which comprises one or moreamino acid sequences of complementarity determining regions (CDRs)defined in the present application and which peptide is capable ofbinding to at least one epitope of human CLEVER-1.

Preferred Embodiments

One embodiment of the present invention is directed to an agent capableof binding to human CLEVER-1 recognizing an epitope of CLEVER-1, whereinthe epitope is discontinuous and comprises the amino acid sequences:

(SEQ ID NO: 1) PFTVLVPSVSSFSSR, and (SEQ ID NO: 2) QEITVTFNQFTK of humanCLEVER-1,

and said agent comprises one or more amino acid sequences ofcomplementarity determining regions (CDRs) binding to said epitopesequences selected from the group consisting of

(SEQ ID NO: 7) TSGMGIG, (SEQ ID NO: 8) HIWWDDDKRYNPALKS, (SEQ ID NO: 9)HYGYDPYYAMDY, (SEQ ID NO: 10) TASSSVSSSYLH, (SEQ ID NO: 11) RTSNLAS, and(SEQ ID NO: 12) HQYHRSPPT.

In some preferred embodiments of the present invention the discontinuousepitope of human CLEVER-1 further comprises one or more of amino acidsequences selected from the group consisting of

(SEQ ID NO: 3) ATQTGRVFLQ, (SEQ ID NO: 4) DSLRDGRLIYLF, (SEQ ID NO: 5)SKGRILTMANQVL, and (SEQ ID NO: 6) LCVYQKPGQAFCTCR.

A part of the target protein human CLEVER-1, i.e. human Stabilin-1, hasdefined in SEQ ID NO: 31. The epitopes SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 on the CLEVER-1corresponds amino acids 420-434, 473-484, 390-399, 576-587, 615-627 and313-327 of target protein human CLEVER-1 defined in SEQ ID NO: 31.

In some preferred embodiments of the present invention the agent capableof binding to an epitope of human CLEVER-1 comprises at least two,preferably three, more preferably four, even more preferably five, andmost preferably all six amino acid sequences of complementaritydetermining regions (CDRs) defined above.

According to the present invention, the agent capable of binding tohuman CLEVER-1 may be selected from the group consisting of an antibody,single chain Fv or Fab fragment(s), peptide(s) or macromolecule(s).

In some preferred embodiments of the present invention an agent capableof binding to human CLEVER-1 is a humanized antibody or single chain Fvor Fab fragment and said antibody or humanized single chain Fv or Fabfragment comprises

a) constant regions of human IgG heavy chain and kappa light chain, and

b) one or more of the following sequences of complementarity determiningregions (CDRs)

i) of the heavy chain CDR 1: (SEQ ID NO: 7) TSGMGIG, and/or CDR 2: (SEQID NO: 8) HIWWDDDKRYNPALKS, and/or CDR 3: (SEQ ID NO: 9) HYGYDPYYAMDY;and ii) of the light chain CDR 1: (SEQ ID NO: 10) TASSSVSSSYLH, and/orCDR 2: (SEQ ID NO: 11) RTSNLAS, and/or CDR 3: (SEQ ID NO: 12) HQYHRSPPT.

The humanized antibody or single chain Fv or Fab fragment capable ofbinding to an epitope of human CLEVER-1 recognizing discontinuousepitope sequences as defined above. The discontinuous epitope of humanCLEVER-1 comprises at least sequences SEQ ID NO: 1 and SEQ ID NO: 2. Insome embodiments the discontinuous epitope of human CLEVER-1 furthercomprises one or more of sequences selected from the group consisting ofSEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

In some embodiments of the present invention referred to above at leasttwo, preferably three, more preferably four, even more preferably five,and most preferably all six CDRs defined above are comprised in thehumanized antibody or single chain Fv or Fab fragment.

In some embodiments of the present invention the human IgG heavy chainvariable region sequence of the humanized antibody or single chain Fv orFab fragment is selected from the group consisting of SEQ ID NO: 14, SEQID NO: 16, SEQ ID NO 18: and SEQ ID NO: 20, preferably SEQ ID NO: 16,SEQ ID NO: 18 and SEQ ID NO: 20. In some embodiments of the presentinvention the human IgG light chain variable region sequence of thehumanized antibody or single chain Fv or Fab fragment is selected fromthe group consisting of SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQID NO: 28 and SEQ ID NO: 30, preferably SEQ ID NO: 30.

In many embodiments of the humanized antibody or single chain Fv or Fabfragment according to the invention the constant regions of the humanIgG heavy chain and kappa light chain are as such. Human IgG4 constantregions are preferred. Many preferred embodiments comprise the humanIgG4 heavy and IgG4 kappa light chain with mutations L248E and/or,preferably and, S241P.

In some embodiments of the present invention the humanized antibody orthe single chain Fv or Fab fragment is capable of binding to humanCLEVER-1 with a relative IC50<1.0, preferably <0.8, more preferably <0.6and most preferably <0.5 in comparison to the IC50 of monoclonalantibody 3-372 (DSM ACC2520 deposited at DSMZ-Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH on Aug. 21, 2001).

According to one embodiment of the invention the combination of thehuman IgG heavy and light chain variable regions are selected from thecombinations presented in Table 5 having capable of binding to humanCLEVER-1 with a relative IC50<1.0 in comparison to the IC50 ofmonoclonal antibody 3-372 (DSM ACC2520 deposited at DSMZ-DeutscheSammlung von Mikroorganismen und Zellkulturen GmbH on Aug. 21, 2001).

A pharmaceutical composition according to the invention comprises theagent capable of binding to human CLEVER-1 or the humanized antibody orthe single chain F_(V) or Fab fragment described above and anappropriate excipient.

A Modulation of Tumour Promoting Macrophages (M2) into Pro-InflammatoryMacrophages (M1)

It has also been found out that an agent capable of binding to humanCLEVER-1, especially to specific epitope sequences on CLEVER-1 definedin the present application, can be used to activate macrophages toswitch their phenotype from M2 macrophages into M1 macrophages.Especially, an agent capable of binding to CLEVER-1 on TAMs can be usedto achieve a modulation of tumour promoting macrophages (M2) intopro-inflammatory macrophages (M1). This modulation increases T-cellactivation and eventually leads e.g. to removal of cancer originatedimmune suppression. More precisely, it has been found out that an agentcapable of binding to specific sequences on CLEVER-1 molecule can beused to remove immune suppression by modulating M2 macrophages into M1macrophages. Consequently, the present finding provides a method foraffecting the immune system in an individual and is especially useful intreating cancer or preventing metastasis, but not limited to thisapproach.

Macrophages may be divided into two distinct phenotypes: M1 and M2macrophages. M1 macrophages are classical pro-inflammatory macrophages,which produce large quantities of pro-inflammatory cytokines andco-stimulatory molecules, and are very efficient in activation of T-cellresponses. M2 macrophages, in contrast, are immune suppressing cells,which synthesize anti-inflammatory cytokines and induce regulatory Tcells and hence profoundly dampen antigen-driven T cell activation.Tumour-associated macrophages (TAMs) are considered harmful as theymature into M2 macrophages (tumour promoting macrophages) within thetumour environment and suppress anti-tumour immune response and mediateangiogenic switch, a crucial step in cancer growth. The M2 macrophagescan be modulated into M1 macrophages (pro-inflammatory macrophages) andsuch phenotype conversion from M2 to M1 may directly or indirectly causetumour rejection.

In the present context the expression “M1 macrophages” or“pro-inflammatory macrophages” refers to the macrophages characterizedby an increased measured level of macrophage/monocyte TNF-alpha (TNF-α)secretion or HLA-DR expression. The modulation of M2 macrophages into M1macrophages will increase monocyte TNF-alpha secretion and also HLA-DRexpression compared to the control values measured before administeringan agent capable of binding to human CLEVER-1 or the values of one ormore previous measurements carried out at different time points in thesame patient. It is important to compare measured values of monocyteTNF-alpha secretion and HLA-DR expression to the values of the samepatient, since the level of these markers may vary from an individual toanother and e.g. cytokines such as interferon-gamma and LPS activationmay increase TNF-alpha expression by the M2 macrophages.

It has surprisingly been found that M2 macrophages can be activated tomodulate M1 macrophages by contacting the said macrophages by an agentcapable of binding to human CLEVER-1, e.g. by an antibody or a fragmentthereof, peptide(s) or macromolecule(s) as defined in the presentapplication. Especially it has been found out that the M2 macrophagesassociated with malignant tumours can be modulated or re-polarized intoM1 macrophages by contacting the said macrophages by an agent capable ofbinding to human CLEVER-1 on TAMs. Both phenotypes may be present atsame time and both of the phenotypes may be found in tumours.

An agent capable of binding to human CLEVER-1, such as an antigen or afragment thereof, peptide(s) or macromolecule(s), is bound to humanCLEVER-1 for achieving said modulation or re-polarization of macrophagephenotypes. It has been identified that the agents specific for CLEVER-1protein recognize a specific CLEVER-1 epitope sequences defined in thepresent application.

A specific binding to two or more said epitope sequences on CLEVER-1 onTAMs will provide a novel method for treating cancers or preventingmetastasis without harmful side-effects since the treatment can betargeted to specific epitopes for achieving desired modulation ofmacrophage phenotype. Consequently, the findings described here areespecially useful in the treatment or prevention of all kinds ofmalignant tumours associated with an increased amount of tumourpromoting macrophages or other pathologies such as chronic inflammationwhere an individual presents a dominance of immune suppression.Consequently, a method for treating cancer or preventing metastasiscomprising administering to an individual an agent capable of binding tohuman CLEVER-1, preferably to specific epitopes on CLEVER-1 moleculedefined above. The method comprises treating or preventing cancer byreducing tumour size and/or; by reducing tumour growth in an individual;and/or by inhibiting cancer cell transmigration and metastasisformation. Thus, any benign or malignant tumour or metastasis ofmalignant tumour, such as skin cancer and colon cancer can be treated.Also leukemias, lymphomas and multiple myelomas can be treated.Particularly, melanomas and lymphomas are expected to respond very wellto the treatment based on animal models.

Macrophages have also an important role during inflammation andinfection resolution besides affecting in the growth or regression oftumours. In infections, a switch from M1 to M2 macrophage can occur,leading to the generation of suppressive environment that abrogateseffector immunity. Consequently, the findings described here to modulatemacrophages phenotype are also useful in the treatment of chronicinfections to remove immune suppression against the infective antigens.The invention also concerns a method for treating chronic infectionscomprising administering to an individual an agent capable of binding toCLEVER-1, preferably to two or more specific epitope sequences onCLEVER-1 molecule defined in the present application, wherein said agentmay activate macrophages to switch their phenotype from M2 into M1.

Further, an agent capable of binding to CLEVER-1 molecule on macrophagesand monocytes in an individual can be used as an adjuvant in vaccines.The said agent achieves re-polarization of macrophages and thus removesor at least decreases immune suppression against the vaccine antigens.Any antigen-induced vaccination may benefit if the host or vaccinationsite can temporally be removed from immune suppressive elements.

The modulation of M2 into M1 macrophages may be verified by measuringmonocyte TNF-alpha secretion from human blood samples. Consequently, theincreased secretion of TNF-alpha may be used as a marker for monitoringtreatment response in an individual. The TNF-alpha secretion may bedetermined from the peripheral blood monocytes enriched from the blooddrawn from a patient. A level of the TNF-alpha measured may be used as amarker for the patient response to the treatment comprisingadministering an agent capable of binding to CLEVER-1 in the patient,when the level is compared to control level measured from the samepatient before administering said agent in the patient, or the values ofone or more previous measurements carried out at different time pointsin the same patient.

A method for estimating of the efficacy of anti-CLEVER-1 therapy bymonitoring a development of a modulation of M2 macrophages into M1macrophages, when an agent capable of binding to CLEVER-1, preferably tosaid one two or more specific epitope sequences on CLEVER-1, isadministered in a patient, comprising the steps of

-   -   (a) obtaining peripheral blood monocytes (PBLs) from a blood        sample drawn from said patient,    -   (b) measuring the TNF-α secretion of said PBLs, and/or    -   (c) measuring HLA-DR expression on CD14 positive PBLs, and    -   (e) comparing values of the TNF-α secretion and/or the HLA-DR        expression measured in steps (b) and (c) to control values for        an estimation of the efficacy of the anti-CLEVER-1 treatment,        wherein the control values are the values measured before        administering an agent capable of binding to CLEVER-1 in the        patient or the values of one or more previous measurements        carried out at different time points in the same patient and        wherein an increased TNF-alpha secretion or HLA-DR expression is        indicative of modulation of M2 macrophages into M1 macrophages.

Determining of TNF-alpha secretion from peripheral blood monocytesobtained from a blood sample drawn from the patient can be carriedcommonly known methods, for example by using a commercial TNF-alphaELISA kit. The HLA-DR expression on CD14 positive monocytes can also bemonitored by using a known method by flow cytometry.

The development of modulation of M2 macrophages into M1 macrophages maybe monitored by comparing a measured level of monocyte TNF-alphasecretion to the control values measured before administering an agentcapable of binding to CLEVER-1 in the patient, or the values of one ormore previous measurements carried out at different time points in thesame patient. For example, a decreased level of monocyte TNF-alphasecretion compared to the results from previous measurements or to acontrol may be used to indicate higher expression of M2 macrophages,while an increased level of TNF-alpha, compared to the results fromprevious measurements or to a control may be used to indicate that moreexpression of M1 macrophages with lower expression of M2 macrophages,wherein it can also be used to indicate the efficacy of theanti-CLEVER-1 treatment. The increased level of TNF-alpha indicates moreexpression of M1 macrophages with lower expression of M2 macrophages,i.e. it attributes responsiveness to said therapy. An agent capable ofbinding to CLEVER-1 will activate at least a part of the M2 macrophagesto re-polarize into M1 macrophages and after the administration of saidagent both macrophage phenotypes may be present, but the increasedexpression of the M1 macrophages may be observed compared to thesituation before the administration of said agent. Typically, at least atwo fold increase of the measured TNF-alpha secretion compared to thecontrol value is indicative of modulation of M2 macrophages into M1macrophages and so to indicate the patient responsiveness to thetherapy.

Diseases Responding to the Treatment

Balancing immune activation and suppression is very critical for thehomeostasis of a human (or animal) in fights against foreign materialborn in or entering the human (or animal). The example of Palani et al.(2016) is a physiological example of this and shows how local immunesuppression is critical for the wellbeing of an embryo in an environmentdominated by a mother's immune defence. The same could take place inchronic infections as some pathogens (e.g. tuberculosis) have learned toutilize a similar hiding mechanism against the host immune system andcould establish chronically infected sites (hepatitis). To remove thislocal immune suppression could help the host to fight against theseinfections as it would do to improve vaccination against these resistantpathogens.

Tumours have adapted this immune suppression to their benefit as well.The method according to the present invention for treating or preventingcancer by reducing the size of malignant tumour; by reducing malignanttumour growth; and/or by inhibiting cancer cell transmigration andmetastasis formation is applicable to all forms of cancers. Thus, anybenign or malignant tumour or metastasis of malignant tumour, such asskin cancer and colon cancer can be treated. Also leukemias, lymphomasand multiple myelomas can be treated. Particularly, melanomas andlymphomas are expected to respond very well to the treatment based onanimal models.

We believe that the agent capable of binding to CLEVER-1 or a humanizedantibody or single chain Fv or Fab fragment according to the presentinvention or the pharmaceutical composition according to the presentinvention is useful in the treatment or prevention of all kinds ofsarcomas, for example fibrosarcoma, liposarcoma, chondrosarcoma,osteosarcoma, angiosarcoma, lymphangisarcoma, leiomyosarcoma, andrhabdomyosarcoma, mesothelioma, meningoma, leukemias, lymphomas, as wellas all kinds of carcinomas, such as squamous cell carcinomas, basal cellcarcinoma, adenocarcinomas, papillary carcinomas, cystadenocarcinomas,bronchogenic carcinomas, melanomas, renal cell carcinomas,hepatocellular carcinoma, transitional cell carcinomas,choriocarcinomas, seminomas, and embryonal carcinomas.

An agent capable of binding to human CLEVER-1 in an individual or ahumanized antibody or single chain Fv or Fab fragment or apharmaceutical composition according to the invention is suitable foruse in removing tumour or antigen induced immunosuppression bymodulating M2 macrophages into M1 macrophages, wherein the agent bindsto an epitope sequences of human CLEVER-1 defined in the presentapplication.

An agent capable of binding to human CLEVER-1 in an individual or ahumanized antibody or single chain Fv or Fab fragment or apharmaceutical composition according to the invention is suitable foruse in treating or preventing cancer by reducing size of malignanttumour; by reducing malignant tumour growth in an individual; and/or byinhibiting cancer cell transmigration and metastasis formation, whereinimmune suppression around the malignant growth is removed by modulatingM2 macrophages into M1 macrophages.

An agent capable of binding to human CLEVER-1 in an individual or ahumanized antibody or single chain Fv or Fab fragment or apharmaceutical composition according to the invention is also suitablefor use in treating chronic infections in an individual, wherein immunesuppression against the infective antigens is removed by modulating M2macrophages into M1 macrophages.

An agent capable of binding to human CLEVER-1 in an individual or ahumanized antibody or single chain Fv or Fab fragment or apharmaceutical composition according to the invention is also suitablefor use as an adjuvant of a vaccine, wherein immune suppression againstvaccine antigens is removed by modulating M2 macrophages into M1macrophages.

A method for modulating M2 macrophages into M1 macrophages comprisesadministering to a subject in need thereof an agent capable of bindingto CLEVER-1, preferably capable of binding to specific sequences onCLEVER-1 molecule as defined in the present application. The said methodcan be used in treatment of cancer or in preventing metastasis in anindividual, or in treatment of chronic infections in an individual. Thetreatment response may be verified by measuring the TNF-α secretion ofsaid PBLs and/or HLA-DR expression on CD14 positive PBLs as described onthe present application.

Administration Routes, Formulations and Required Dose

The pharmaceutical compositions to be used in the present invention canbe administered by any means that achieve their intended purpose. Forexample, administration can be intravenous, intraarticular,intra-tumoural or subcutaneous. In addition to the pharmacologicallyactive compounds, the pharmaceutical preparations of the compoundspreferably contain suitable pharmaceutically acceptable carrierscomprising excipients and auxiliaries that facilitate processing of theactive compounds into preparations that can be used pharmaceutically.

The dose chosen should be therapeutically effective with regard to thedisease treated. Accordingly immunosuppression should be sufficient totreat the disease without effects essentially endangering the soughtoutcome of the treatment. When treating or preventing cancer the doseshould be sufficient to reduce size of malignant tumour, reducemalignant tumour growth and/or inhibit cancer cell transmigration andmetastasis formation. The dose is dependent on the turnover of theadministered agent but typically these treatments follow a regimen of 1to 5 mg/kg every other 2 to 4 weeks.

EXAMPLES

The following experimental section illustrates the invention byproviding examples.

The examples 1 to 10 illustrate discontinuous epitope mapping of humanCLEVER-1 and generation of humanized antibodies from the 3-372 mousemonoclonal antibody (DSM ACC2520 deposited at DSMZ-Deutsche Sammlung vonMikroorganismen and Zellkulturen GmbH on Aug. 21, 2001) using CompositeHuman Antibody™ technology. Anti-CLEVER-1 antibodies ability to promoteimmune activation is illustrated in Examples 11 to 14.

Example 1 illustrates full discontinuous epitope mapping of antibodies,which target human CLEVER-1.

Examples 2 to 6 illustrate determination of heavy and light chain Vregion (V_(H) and V_(K)) sequences of the anti-Clever 1 antibody clone3-372 and production of chimeric antibodies comprising 3-372 variableregions and human IgG4 heavy chain and kappa light chain constantregions. mRNA was extracted from hybridoma clone 3-372, reversetranscribed, PCR amplified and antibody-specific transcripts werecloned. The nucleotide and amino acid sequences of the antibody heavyand light chain variable regions were determined, and an analysis of thesequence data was performed for humanization using Antitope'sproprietary Composite Human Antibody™ technology.

Examples 2 to 6 demonstrate that: Variable regions from the 3-372 mouseanti-Clever 1 antibody have been cloned and sequenced. Variable regiongenes have been combined with human IgG4(S241P) heavy chain and kappalight chain constant regions and expressed in NSO cells to produce achimeric anti-Clever 1 antibody. A competition ELISA assay fromNSO-derived chimeric antibody was used to demonstrate that the bindingefficiency of the chimeric antibody for Clever-1 is similar to that ofthe parental murine antibody.

Examples 7 to 10 illustrate: Design of anti-CLEVER-1 Composite HumanAntibodies™ which were expressed and tested for binding to humanClever-1. Key residues involved in the structure and binding ofanti-CLEVER-1 were determined by structure and homology modelling togenerate a ‘constraining residue map’. The constraining residue map wasused as a template to source segments of human V region sequence fromdatabases containing unrelated human antibody sequences. Each selectedsequence segment, as well as the junctions between segments, were testedfor the presence of potential T cell epitopes using in silico (iTope™and TCED™) analysis. Using this method, all Composite Human Antibody™sequence variants were designed to avoid T cell epitopes. CompositeHuman Antibody™ V region genes were generated using syntheticoligonucleotides encoding combinations of the selected human sequencesegments. These were then cloned into vectors containing humanIgG4(S241P) heavy chain and kappa light chain constant regions, andantibodies were produced and tested for binding to target antigen bycompetition ELISA in comparison to the original reference murinemonoclonal antibody.

Examples 7 to 10 demonstrate construction of four VH and five VκComposite Human Antibody™ V regions. Combinations of composite heavy andlight chains were expressed in NSO cells, purified and tested forbinding to CLEVER-1 in a competition ELISA assay. The resultsdemonstrated that the binding efficiency of many of the Composite HumanAntibodies™ to CLEVER-1 was at least as good as that of the chimericreference antibody and several were markedly better. Based on theabsence of a potential glycosylation site and an unpaired cysteine, andexpression and binding efficiency data sets generated, three potentialleads were designated as follows: VH2/VK5, VH3/VK5, and VH4/VK5.

Examples 11 to 12 illustrate anti-CLEVER-1 antibody binding on humanperipheral blood monocytes and activating TNF-alpha secretion on humanperipheral blood monocytes. Example 13 illustrates the mode of action ofanti-CLEVER-1-antibodies on tumor-associated macrophages in mousesyngeneic cancer models.

Example 14 illustrates that CLEVER-1 ligation with 9-11 and 3-372antibodies promotes opposing effects on mTOR and c-Jun signaling inhuman peripheral blood monocytes.

Example 1

Full Discontinuous Epitope Mapping of Human CLEVER-1

Tentative discontinuous epitopes for four antibodies were establishedemploying Pepscan analysis. Antibodies FAR02 VH3/VK5 and FU-HI-3-372target same discontinuous epitope, while antibodies 3-266 and AK FUMM9-11 target other distinct epitopes. The study was conducted at PepscanPresto BV, (Zuidersluisweg 2, 8243RC Lelystad, The Netherlands).

The antibodies 3-266, FAR02 VH3/VK5, FU-HI-3-372 and AK FUMM 9-11 wereprovided by Faron Pharmaceuticals Oy.

The target protein human CLEVER-1, i.e. human Stabilin-1, was defined bySEQ ID NO: 31. Disulfide bridges connect residues (numbering UniprotSTAB1_HUMAN):

112 126 120 136 138 147 160 171 164 181 183 192 199 210 204 217 236 247241 257 259 270 732 746 740 756 758 767 822 837 831 846 865 879 873 889891 902 908 922 916 932 934 945 951 964 958 974

CLIPS Technology

The CLIPS technology employed structurally fixes peptides into definedthree-dimensional structures. This results in functional mimics of eventhe most complex binding sites. CLIPS technology is now routinely usedto shape peptide libraries into single, double or triple loopedstructures as well as sheet- and helix-like folds. The CLIPS reactiontakes place between bromo groups of the CLIPS scaffold and thiolsidechains of cysteines. The reaction is fast and specific under mildconditions. Using this elegant chemistry, native protein sequences aretransformed into CLIPS constructs with a range of structures. (Timmermanet al., J. Mol. Recognit. 2007; 20: 283-29)

CLIPS library screening starts with the conversion of the target proteininto a library of up to 10,000 overlapping peptide constructs, using acombinatorial matrix design. On a solid carrier, a matrix of linearpeptides is synthesized, which are subsequently shaped into spatiallydefined CLIPS constructs. Constructs representing both parts of thediscontinuous epitope in the correct conformation bind the antibody withhigh affinity, which is detected and quantified. Constructs presentingthe incomplete epitope bind the antibody with lower affinity, whereasconstructs not containing the epitope do not bind at all. Affinityinformation is used in iterative screens to define the sequence andconformation of epitopes in detail. The target protein containing adiscontinuous conformational epitope is converted into a matrix library.Combinatorial peptides are synthesized on a proprietary minicard andchemically converted into spatially defined CLIPS constructs.

Heat Map Analysis

A heat map is a graphical representation of data where the values takenby a variable in a two-dimensional map are represented as colors.

For double-looped CLIPS peptides, such a two-dimensional map can bederived from the independent sequences of the first and second loops.For example, sequences of the 16 CLIPS peptides are effectivelypermutations of e.g. 4 unique sub-sequences in e.g. loop 1 and e.g. 4unique sub-sequences in e.g. loop 2. Thus, observed ELISA data can beplotted in a 4×4 matrix, where each X coordinate corresponds to thesequence of the first loop, and each Y coordinate corresponds to thesequence of the second loop.

To further facilitate the visualization, ELISA values can be replacedwith colours from a continuous gradient. For example extremely lowvalues can be coloured in green, extremely high values in coloured inred, and average values are coloured in black.

Synthesis of Peptides

To reconstruct epitopes of the target molecule a library of peptides wassynthesized. An amino functionalized polypropylene support was obtainedby grafting with a proprietary hydrophilic polymer formulation, followedby reaction with t-butyloxycarbonyl-hexamethylenediamine (BocHMDA) usingdicyclohexylcarbodiimide (DCC) with Nhydroxybenzotriazole (HOBt) andsubsequent cleavage of the Boc-groups using trifluoroacetic acid (TFA).Standard Fmoc-peptide synthesis was used to synthesize peptides on theamino-functionalized solid support by custom modified JANUS liquidhandling stations (Perkin Elmer).

Synthesis of structural mimics was done using Pepscan's proprietaryChemically Linked Peptides on Scaffolds (CLIPS) technology. CLIPStechnology allows to structure peptides into single loops, double-loops,triple loops, sheet-like folds, helix-like folds and combinationsthereof. CLIPS templates are coupled to cysteine residues. Theside-chains of multiple cysteines in the peptides are coupled to one ortwo CLIPS templates. For example, a 0.5 mM solution of the P2 CLIPS(2,6-bis(bromomethyl)pyridine) is dissolved in ammonium bicarbonate (20mM, pH 7.8)/acetonitrile (1:3(v/v)). This solution is added onto thepeptide arrays. The CLIPS template will bind to side-chains of twocysteines as present in the solid-phase bound peptides of thepeptide-arrays (455 wells plate with 3 μl wells). The peptide arrays aregently shaken in the solution for 30 to 60 minutes while completelycovered in solution. Finally, the peptide arrays are washed extensivelywith excess of H₂O and sonicated in disrupt-buffer containing 1% SDS/0.1beta-mercaptoethanol in PBS (pH 7.2) at 70° C. for 30 minutes, followedby sonication in H₂O for another 45 minutes. The T3 CLIPS carryingpeptides were made in a similar way but now with three cysteines.

ELISA Screening

The binding of antibody to each of the synthesized peptides was testedin a PEPSCAN-based ELISA. The peptide arrays were incubated with primaryantibody solution (overnight at 4° C.). After washing, the peptidearrays were incubated with a 1/1000 dilution of an appropriate antibodyperoxidase conjugate (SBA; Table 1) for one hour at 25° C. Afterwashing, the peroxidase substrate 2,2′-azino-di-3-ethylbenzthiazolinesulfonate (ABTS) and 20 μl/ml of 3 percent H₂O₂ were added. After onehour, the colour development was measured. The colour development wasquantified with a charge coupled device (CCD)—camera and an imageprocessing system.

TABLE 1 Details of the antibodies Name Supplier Cat. No goat anti-humanHRP conjugate Southern Biotech 2010-05 rabbit anti-mouse IgG (J + L) HRPconjugate Southern Biotech 6175-05 goat anti-rat IgM + IgG (H + L) HRPSouthern Biotech 3010-05 conjugate

Langedijk et al. (2011). Helical peptide arrays for lead identificationand interaction site mapping, Analytical Biochemistry 417: 149-155

Design of Peptides

Different sets of peptides were synthesized according to the followingdesigns. Note that in some sets peptides were synthesized in a randomorder. Below the actual peptide order is shown.

Set 1

Mimic Type linear Label LIN Description Linear 15-mer peptides derivedfrom the target sequence of human Clever-1 with an offset of one residue. Sequences (first 10)QVLFKGCDVKTTFVT VLFKGCDVKTTFVTH LFKGCDVKTTFVTHV FKGCDVKTTFVTHVPKGCDVKTTFVTHVPC GCDVKTTFVTHVPCT CDVKTTFVTHVPCTS DVKTTFVTHVPCTSCVKTTFVTHVPCTSCA KTTFVTHVPCTSCAA

Set 2

Mimic Type linear Label LIN.AA DescriptionPeptides of set 1, but with residueson positions 10 and 11 replaced by Ala.Once a native Ala would occur on either position, it is replaced by Gly.Sequences (first 10) GAETPCNGHAACLDG LTMANQVLAAAISEE ILLPPTILPAAPKHCDRNGTCVCQAAFRGS PGYTQQGSEAAAPNP PIDPCRAGNAACHGL HTDALCSYVAAGQSRKGCDVKTTFAAHVPC CQALNTSTCAANSVK RAVGGGQRVAACPPG

Set 3

Mimic Type linear Label LIN20.C DescriptionLinear peptides of length 20 derived from the target sequence of humanClever-1 with an offset of one residue. Cys residues are protected byacetimidomethyl (Acm, denoted “2”). Sequences (first 10)2H2PENYHGDGMV2LPKDP2 SGWLRELPDQITQD2RYEVQ LAQH2HLHAR2VSQEGVAR2IKKQT2PSGWLRELPDQITQ 2RESEVGDGRA2YGHLLHEV QRV2T2PPGFGGDGFS2YGDNGVFHVVTGLRWQAPSGTPG AT2QVTADGKTS2V2RESEV KYSYKYKDQPQQTFNIYKAN2VYIHDPTGLNVLKKG2ASY

Set 4

Mimic Type linear Label LIN25.C DescriptionLinear peptides of length 25 derived from the target sequence of humanClever-1 with an offset of one residue. Cys residues are protectedby Acm (“2”). Sequences (first 10) KKG2ASY2NQTIMEQG22KGFFGPDPD2QSV2S2VHGV2NHGPRGDGS2L GPGQSR2T2KLGFAGDGYQ2SPIDPIFPKE2VYIHDPTGLNVLKKG2ASY PTILPILPKH2SEEQHKIVAGS2VDENFRGSA2QE2QDPNRFGPD2QSV2 QNTQ2SAEAPS2R2LPGYTQQGSE2GRV2VAIDE2ELDMRGG2HTDAL2S APSGTPGDPKRTIGQILASTEAFSRDGMV2LPKDP2TDNLGG2PSNSTL2

Set 5

Mimic Type Constrained peptides, mP2 CLIPS Label LOOP DescriptionPeptides of length 17. On positions2-16 are 15-mer sequences derived fromthe target protein. On positions 1 and 17 are Cys residues joined bymP2 CLIPS. Native Cys residues are protected by Acm (“2”). Sequences(first 10) CL2SYVGPGQSR2T2KC C2SYVGPGQSR2T2KLC CSYVGPGQSR2T2KLGCCYVGPGQSR2T2KLGFC CVGPGQSR2T2KLGFAC CGPGQSR2T2KLGFAGC CPGQSR2T2KLGFAGDCCGQSR2T2KLGFAGDGC CQSR2T2KLGFAGDGYC CSR2T2KLGFAGDGYQC

Set 6

Mimic Type Linear disulphide mimics Label CYS22 DescriptionLinear disulphide mimics of length 22designed based on Uniprot information on disulphide bridges for humanCLEVER-1. Cys residues within a mimic,that do not participate in disulphide bridge formation, are protected byAcm (“2”). Sequences (first 10) WGSR2HECPGGAETP2NGHGTCSR2HECPGGAETP2NGHGTCLD 2HECPGGAETP2NGHGTCLDGM ECPGGAETP2NGHGTCLDGMDRGAETPCNGHGT2LDGMDRNGTC ETPCNGHGT2LDGMDRNGTCV2 PCNGHGT2LDGMDRNGTCV2QELDGMDRNGT2VCQENFRGSACQ GMDRNGT2VCQENFRGSACQE2 DRNGT2VCQENFRGSACQE2QD

Set 7

Mimic Type Combinatorial disulphide bridge mimics Label CYS27Description Combinatorial peptides of length 27.On positions 1-11 and 16-27 are 11-mer sequences derived from the targetsequence on page 7 joined by “GGSGG” linker. This peptideset was designed based on disulphide bridge information obtained fromUniprot. Cys residues within a mimic,that do not participate in disulphide bridge formation, are protected byAcm (“2”). Sequences (first 10) PGYWGSR2HECGGSGGAETP2NGHGTCYWGSR2HECPGGGSGGAETP2NGHGTC GSR2HECPGGAGGSGGAETP2NGHGTCR2HECPGGAETGGSGGAETP2NGHGTC HECPGGAETP2GGSGGAETP2NGHGTCCPGGAETP2NGGGSGGAETP2NGHGTC PGYWGSR2HECGGSGGTP2NGHGTCLDYWGSR2HECPGGGSGGTP2NGHGTCLD GSR2HECPGGAGGSGGTP2NGHGTCLDR2HECPGGAETGGSGGTP2NGHGTCLD

Set 8

Mimic Type Discontinuous matrix, T3 CLIPS Label MAT DescriptionPeptides of length 33. On positions 2-16 and 18-32 are 15-mer peptidesderived from the target sequence ofhuman Clever-1. On positions 1, 17 and33 are Cys residues joined by T3 CLIPS. Native Cys residues areprotected by Acm (“2”). Sequences (first 10)CPNRFGPD2QSV2S2VCV2S2VHGV2NHGPRGC CHGDGMV2LPKDP2TDCSAG2FAF2SPFS2DRCC2VD2QALNTST2PPNCPKH2SEEQHKIVAGSC CPKH2SEEQHKIVAGSCGPD2TQ2PGGFSNP2CCRYEVQLGGSMVSMSGCVP2TS2AAIKKQT2PC CHKIVAGS2VD2QALNCIHMLDGILLPPTILPCCF2T2RPGLVSINSNACVTADGKTS2V2RESEC C2VYIHDPTGLNVLKKCGSGGV2QQGT2APGFCCLRVAVAMMDQG2REICDGRA2YGHLLHEVQKC CYSYKYKDQPQQTFNICHEVQKATQTGRVFLQC

Screening Details

Antibody binding depends on a combination of factors, includingconcentration of the antibody and the amounts and nature of competingproteins in the ELISA buffer. Also, the pre-coat conditions (thespecific treatment of the peptide arrays prior to incubation with theexperimental sample) affect binding. These details are summed up inTable 2. For the Pepscan Buffer and Preconditioning (SQ), the numbersindicate the relative amount of competing protein (a combination ofhorse serum and ovalbumin).

TABLE 2 Screening conditions Label Dilution Sample bufferPre-conditioning 3-266 5 μg/ml 10% SQ 10% SQ AK FUMM 9-11 1 μg/ml 50% SQ50% SQ FAR02 VH3/VK5 3 μg/ml  1% SQ  1% SQ FU-HI_3-372 5 μg/ml  1% SQ 1% SQ

Results

Antibody 3-266

When tested under high stringency conditions antibody 3-266 did not bindany peptides present on the arrays. When tested under low stringencyconditions the antibody bound peptides from all sets. Results obtainedwith simple epitope mimics suggest that sequence ₁₀₃₀QWLKSAGITLPADRR₁₀₄₄represents the dominant part of the epitope. Data obtained withcombinatorial epitope mimics (FIG. 1) suggest that the antibodyadditionally recognizes sequence ₈₅₇LHARCVSQEGVARCR₈₇₁. Moreover, a weakand consistent signal was recorded for peptides with sequence₄₃₅TMNASLAQQLCRQHI₄₅₀. FIG. 1a illustrates heatmap representation ofresults obtained for antibody 3-266 on set 8 (discontinuous epitopemimics). Average signal is plotted in black and extremely high signal isplotted in light. Boxed regions are magnified.

Antibody AK FUMM 9-11

When tested under high stringency conditions antibody AK-FUMM 9-11 boundpeptides from all sets. Results obtained with simple epitope mimicssuggest that the antibody ₈₈₅PSNPCSHPDRGG₈₉₆, which represents thedominant part of epitope. Data obtained with combinatorial epitopemimics suggest that the antibody additionally recognizes combinatorialepitope mimics containing sequence ₁₆₆FRGSACQECQDPNRF₁₈₀ (FIG. 1b ).FIG. 1b illustrates heatmap representation of results obtained forantibody AK FUMM 9-11 on set 8 (discontinuous epitope mimics). Averagesignal is plotted in black and extremely high signal is plotted inlight. Boxed regions are magnified.

Antibody FAR02 VH3A/K5 and FU-H1:3-372

When tested under high stringency conditions antibodies FAR02 VH3/vk5and FU-HI-3-372 did not bind any peptides present on the arrays. Whentested under low stringency conditions these antibodies specificallybound peptides only from set 8 (FIG. 2). Analysis of results obtainedwith discontinuous mimics suggests that the antibody recognizes adiscontinuous epitope composed of sequences ₃₉₀ATQTGRVFLQ₃₉₉ (SEQ ID NO:3), ₄₂₀PFTVLVPSVSSFSSR₄₃₄ (SEQ ID NO: 1), ₄₇₃QEITVTFNQFTK₄₈₄ (SEQ ID NO:2), ₅₇₆DSLRDGRLIYLF₅₈₇ (SEQ ID NO: 4), ₆₁₅SKGRILTMANQVL₆₂₇ (SEQ ID NO:5), where sequences ₄₇₃QEITVTFNQFTK₄₈₄ (SEQ ID NO: 2) and₄₂₀PFTVLVPSVSSFSSR₄₃₄ (SEQ ID NO: 1) appear to represent core epitopes.Additional weaker signal was recorded for discontinuous mimicscontaining sequence ₃₁₃LCVYQKPGQAFCTCR₃₂₇ (SEQ ID NO: 6). Resultsobtained with simple epitope mimics do not allow epitope calling. FIG. 2illustrates heatmap representation of results obtained for antibodyFU-HI-3-372 on set 8 (discontinuous epitope mimics). Average signal isplotted in black and extremely high signal is plotted in light. Boxedregions are magnified.

Conclusions

The antibodies were tested against Pepscan peptide arrays. It waspossible to identify tentative discontinuous epitopes for all monoclonalantibodies. Peptide sequences comprising epitopes are listed in Table 3.Antibodies 3-266 and AK FUMM 9-11 bind distinct discontinuous epitopes.Antibodies FAR02 VH3/VK5 and FU-HI-3-372 essentially displayed highlysimilar binding patterns when tested on the arrays and, therefore, wereshown to recognize the same discontinuous epitope in the FAS1/FAS2domains.

TABLE 3 Epitopes found Antibody Epitope sequences Domain 3-266 ₁₀₃₀QWLKSAGITLPADRR ₁₀₄₄ FAS 3 ₈₅₇LHARCVSQEGVARCR₈₇₁ EGF-like 6₄₃₅TMNASLAQQLCRQHI₄₅₀ FAS 1 AK FUMM 9-11 ₈₈₅ PSNPCSHPDRGG ₈₉₆ EGF-like 6₁₆₆ FRGSACQECQDPNRF ₁₈₀ EGF-like 1 FAR02 ₄₂₀ PFTVLVPSVSSFSSR ₄₃₄ FAS 1VH3/VK5 + ₄₇₃ QEITVTFNQFTK ₄₈₄ FAS 1 FU-HI-3-372 ₃₉₀ATQTGRVFLQ₃₉₉ FAS 1₅₇₆DSLRDGRLIYLF₅₈₇ FAS 2 ₆₁₅SKGRILTMANQVL₆₂₇ FAS 2

To compare visually tentative epitopes identified for the abovementioned antibodies a schematic drawing in FIG. 3 was used. Thisschematic was adapted from FIG. 1 from Kzhyshkowska,TheScientificWorldJOURNAL (2010) 10, 2039-2053 representing Stabilin-1(CLEVER-1) domain organization. FIG. 3 illustrates schematically thedomain organization of CLEVER-1 (aa_25-1027 as per target sequence).Arrowheads indicate relative positions of identified binding motifs.Circulated arrowheads indicate positions of dominant epitope cores.

Example 2

mRNA Extraction, RT-PCR and Cloning

mRNA was successfully extracted from the hybridoma cells (PolyA Tractsystem, Promega Cat. No. Z5400). RT-PCR was performed using degenerateprimer pools for murine signal sequences with a single constant regionprimer. Heavy chain variable region mRNA was amplified using a set ofsix degenerate primer pools (HA to HF) and light chain variable regionmRNA was amplified using a set of eight degenerate primer pools (κA toκG and λA). Amplification products were obtained with the heavy chainprimer pool HD and light chain primer pools κB, κC and κG confirming thelight chain is from the K cluster (FIG. 4). Each product was cloned andseveral clones from each sequenced.

Using this methodology, a single VH sequence [SEQ ID NO: 32 (basesequence) and NO: 33 (amino acid sequence)] was identified in pool HDand a single functional Vκ sequence [SEQ ID NO: 34 (base sequence) andNO: 35 (amino acid sequence)] was identified in primer pool κG. The CDRsof the heavy chain stretch from base 91 to 111 (SEQ ID NO: 7), 154 to210 (SEQ ID NO: 8) and 298 to 333 (SEQ ID NO: 9); and the CDRs of thelight chain stretch from base 70 to 105 (SEQ ID NO: 10), 151 to 171 (SEQID NO: 11) and 268 to 294 (SEQ ID NO: 12). CDR definitions and proteinsequence numbering are according to Kabat. An aberrant K light chaintranscript normally associated with the hybridoma fusion partner SP2/0(GenBank M35669) was also identified in primer pools κB and κC.

FIG. 4 illustrates 1% agarose gel separation of hybridoma 3-372 RT-PCRproducts. Gel was stained with SYBR® Green dye (Invitrogen Cat. No.S-7567) and photographed over ultraviolet light. Size marker isGeneRuler™ 1 Kb Plus (Fermentas Cat. No. SM1331). Boxes indicate bandsthat were isolated for cloning and sequencing.

Example 3

Sequence Analysis

The analysis of the sequences obtained from hybridoma expressing 3-372is summarised in Table 4.

TABLE 4 3-372 Antibody Sequence Analysis^(a) H-Chain L-Chain CDR 1Length 7aa 12aa CDR 2 Length 16aa 7aa CDR 3 Length 12aa 9aa ClosestHuman IGHV2-5*10 (73%) IGKV3D-20*01 (65%) Germline^(b) Closest HumanFW1^(b) IGHV2-70*06 (73%) IGKV1D-17*01 (68%) Closest Human FW2^(b)IGHV2-5*09 (86%) IGKV1D-39*01 (73%) Closest Human FW3^(b) IGHV2-70*13(72%) IGKV1D-43*01 (78%) Closest Human J^(b) IGHJ1 (91%) IGKJ4 (80%)^(a)CDR definitions and sequence numbering according to Kabat^(b)Germline ID(s) indicated followed by % homology

Structure and homology analysis of the murine 3-372 variable domainsequence identified four framework residues in the heavy chain variableregion and five framework residues in the light chain variable regionwhich were considered to be critical or possibly important to antigenbinding (“constraining residues”). Additional sequence database analysisrevealed that human framework segments can be found to include alldesirable constraining residues and all CDR residues, thus permittingthe construction of Composite Human Antibodies™.

It was also noted that the 3-372 V-regions contain some unusualfeatures. The VH chain contains a N-glycosylation site at the beginningof CDR1 since residue 30 is N and 32 is S (N-glycosylation signal is NXSor NXT, where X can be any amino acid). Due to the likely exposure ofthis motif on the surface of the antibody, it is probable that this sitewill be glycosylated. Therefore it would be advantageous (if notinvolved in antigen binding) to avoid this site in the Composite HumanAntibodies in order to avoid any manufacturing issues in the future. TheVκ chain also contains a glycosylation site but only in the context of ahuman K constant region since the final amino acid of the Vκ domain isasparagine. The mouse K constant domain begins RA, whereas the human Kconstant domain begins RT, thus forming a glycosylation signal.Therefore sequences for Composite Human Antibodies will be selected toavoid this asparagine.

The K domain also contains an unpaired cysteine at position 47.Molecular modelling suggest that this residue will be buried within thestructure and therefore not available for disulphide bonding; however itcould be a key residue for maintaining the conformation of Vκ CDR2, andtherefore sequences for Composite Human Antibodies will be selected withand without this residue (the latter including the consensus human L atthis position) in order to investigate its effects on antigen binding.

Example 4

Expression of Chimeric Antibody

The 3-372 variable regions were transferred to Antitope's expressionvector system for IgG4(S241P) heavy chain and kappa light chain. NSOcells were transfected via electroporation and selected usingmethotrexate (MTX). A number of MTX resistant colonies were identifiedusing an Fc capture/kappa chain detection ELISA, and cell lines positivefor IgG expression were expanded continuously from 96-well platesthrough to T175 flasks in media containing gradually increasingconcentrations of MTX and subsequently frozen under liquid nitrogen. Ateach stage, IgG expression was quantified.

Chimeric 3-372 IgG4 was purified from cell culture supernatants on aProtein-A Sepharose column and quantified by OD280 nm using anextinction coefficient (Ec(0.1%)) value of 1.55 based on the predictedamino acid sequence for chimeric IgG4. Approximately 90 μg of antibodywas purified and a sample was analysed by reducing SDS-PAGE (FIG. 5).Bands corresponding to the predicted sizes of the heavy and light chainswere observed with no evidence of any contamination; however it wasnotable that the chimeric light chain appears to be glycosylated asevidenced by the greater apparent molecular weight than the mouse lightchain. The heavy chain also appeared to be running slower than is usual,suggesting that it is also N-glycosylated; however digestion withglycosidases would be required to demonstrate that this is indeed thecase.

FIG. 5 illustrates Coomassie Blue-stained SDS-PAGE gel of proteinA-purified chimeric 3-372 IgG4. 1 μg of sample was loaded on a NuPage4-12% Bis-Tris gel (Invitrogen Cat. No. NP0322BOX) and run at 200 V for30 min. Lanes 1&4: Prestained protein standard (Fermentas PageRuler Cat.No. SM1811). Lane 2: 1.0 μg chimeric 3-372 IgG4 antibody. Lane 3 1.0 μgmurine 3-372 antibody.

Example 5

Binding of Chimeric Antibody to CLEVER-1

The binding of NSO derived chimeric 3-372 to CLEVER-1 was assessed bycompetition ELISA. Briefly, a Nunc Immulon 96 well maxisorp plate(Fisher Cat. No. DIS-971-030J) was coated with CLEVER-1 at 1 μg/ml inPBS (100 μl/well) overnight at 4° C., with an additional 1 hour at 37°C. the following morning. Wells were washed with PBS/0.1% Tween 20 andthen blocked for 45 min at room temperature, in 1% Marvel/1% BSA/PBS.

Dilution series of both the chimeric 3-372 and the reference mouse 3-372(5-0.078 μg/ml) were premixed with a constant concentration (0.6 μg/ml)of biotinylated mouse 3-372 antibody in 2% BSA/PBS. The blocked ELISAplate was washed as before and 100 μl of the premixed antibodies addedto each well. The plate was incubated for 1 hour at room temperature.Binding of the biotinylated mouse 3-372 to CLEVER-1 was detected usingStreptavidin-HRP (Sigma Cat. No. S5512) and TMB single solutionsubstrate (Invitrogen Cat. No. 00-2023). The reaction was stopped with3M HCl, absorbance read at 450 nm on a Dynex Technologies MRX TC IIplate reader and the binding curve of the chimeric 3-372 compared tothat of the reference mouse 3-372 antibody (FIG. 6).

FIG. 6 shows the binding profile of the mouse and chimeric antibodiesfor CLEVER-1 in competition with the biotinylated mouse antibody. Thecurves are almost identical giving IC50 values of 0.89 μg/ml for thechimeric antibody compared to 0.77 μg/ml for the mouse antibody. Thisconfirms that the correct variable region sequences have been identifiedand expressed in the chimeric antibody.

Example 7

Design of Composite Human Antibody™ Variable Region Sequences andVariants

Structural models of the murine anti-CLEVER-1 antibody V regions wereproduced using Swiss PDB and analysed in order to identify important“constraining” amino acids in the V regions that were likely to beessential for the binding properties of the antibody. Residues containedwithin the CDRs (using both Kabat and Chothia definitions) together witha number of framework residues were considered to be important. Both theVH and Vκ sequences of anti-Clever 1 contain typical framework residuesand the CDR 1, 2 and 3 motifs are comparable to many murine antibodies.However, we identified a potential site for N-linked glycosylation inthe VH sequence (30N), and an unpaired cysteine in the Vκ sequence(47C).

From the above analysis, it was considered that composite humansequences of anti-CLEVER-1 could be created with a wide latitude ofalternatives outside of the CDRs but with only a narrow menu of possiblealternative residues within the CDR sequences. Preliminary analysisindicated that corresponding sequence segments from several humanantibodies could be combined to create CDRs similar or identical tothose in the murine sequences. For regions outside of and flanking theCDRs, a wide selection of human sequence segments were identified aspossible components of the novel Composite Human Antibody™ V regions.

Based upon the above analysis, a large preliminary set of sequencesegments that could be used to create anti-CLEVER-1 Composite HumanAntibody™ variants were selected and analysed using iTope™ technologyfor in silico analysis of peptide binding to human MHC class II alleles(Perry et al 2008), and using the TCED™ (T Cell Epitope Database) ofknown antibody sequence-related T cell epitopes (Bryson et al 2010).Sequence segments that were identified as significant non-human germlinebinders to human MHC class II or that scored significant hits againstthe TCED™ were discarded. This resulted in a reduced set of segments,and combinations of these were again analysed, as above, to ensure thatthe junctions between segments did not contain potential T cellepitopes. Selected segments were then combined to produce heavy andlight chain V region sequences for synthesis. For anti-CLEVER-1, four VHchains, VH1, VH2; VH3 and VH4 [SEQ ID NO: 13, 15, 17 and 19 (basesequence) and NO: 14, 16, 18 and 20 (amino acid sequence), respectively]and five Vκ chains, VK1, VK2, VK3, VK4 and VK5 [SEQ ID NO: 21, 23, 25,27 and 29 (base sequence) and NO: 22, 24, 26, 28 and 30 (amino acidsequence), respectively] were designed. The heavy chain CDRs VH1, VH2,VH3 and VH4 stretch from base 91 to 111, 154 to 201 and 298 to 333; andthe light chain CDRs VK1, VK2, VK3, VK4 and VK5 stretch from base 70 to105, 151 to 171 and 268 to 294. CDR definitions and protein sequencenumbering are according to Kabat. Of note, three of the VH chains havethe potential N-linked glycosylation site removed (VH2, VH3, and VH4),and two of the Vκ chains have the unpaired cysteine removed (VK4 andVK5).

Example 8

Construction of Composite Human Antibody™ Variants

All variant Composite Human Antibody™ VH and Vκ region genes foranti-Clever 1 were synthesized using a series of overlappingoligonucleotides that were annealed, ligated and PCR amplified to givefull length synthetic V regions. The assembled variants were then cloneddirectly into Antitope's pANT expression vector system for IgG4(S241P)VH chains and Vκ chains (FIG. 7). The VH region was cloned using MluIand HindIII sites, and the Vκ region was cloned using BssHII and BamHIrestriction sites. All constructs were confirmed by sequencing.

FIG. 7 shows the Antitope pANT vector diagram. Both Vh and Vκ vectorscontain genomic DNA fragments incorporating introns and poly Asequences. Expression of both chains is driven by a CMV promoter andselection (on the heavy chain vector) is via a DHFR mini gene.

Example 9

Construction, Expression and Purification of Antibodies

All combinations of composite IgG4(S241P) VH and Vκ chains (i.e. a totalof 20 pairings) were stably transfected into NSO cells viaelectroporation. The stable transfections were selected using 200 nMmethotrexate (MTX) (Sigma cat. no. M8407), methotrexate-resistantcolonies for each construct were tested for IgG expression levels usingan IgG4 ELISA, and the best expressing lines were selected, expanded andfrozen under liquid nitrogen. Successful transfection and stable cloneselection were achieved for all variants except VH3/VK3 and VH4/VK3.

The composite variants of anti-CLEVER-1 were purified from cell culturesupernatants on a Protein A sepharose column (GE Healthcare cat. no.110034-93), buffer exchanged into a PBS and quantified by OD280 nm usingan extinction coefficient (Ec (0.1%)=1.55) based on the predicted aminoacid sequence. The lead Composite Human Antibody™ variants were analysedby reducing SDS-PAGE. Bands corresponding to the predicted sizes of theVH and Vκ chains were observed with no evidence of any contamination(FIG. 8).

FIG. 8 illustrates Coomassie Blue-stained SDS-PAGE gel of selectedprotein A-purified antibodies. 2 μg of each sample was loaded on aNuPage 4-12% Bis-Tris gel (Invitrogen cat. no. NP0322BOX) and run at 200V for 35 min. Size marker is prestained protein standard FermentasPageRuler (cat. no. SM1811).

Example 10

Binding of Composite Human Antibodies™ to CLEVER-1

The binding of NSO derived Composite 3-372 antibodies to CLEVER-1 wasassessed by competition ELISA. Dilution series of the chimeric and thecomposite 3-372 antibodies (5-0.078 μg/ml) were premixed with a constantconcentration (0.6 μg/ml) of biotinylated Mouse 3-372 antibody. Thesewere incubated for 1 hour at room temperature on a 96 well Immulonmaxisorp plate (Fisher Cat. No. DIS-971-030J) precoated with 1 μg/mlCLEVER-1. Binding of the biotinylated Mouse 3-372 to CLEVER-1 wasdetected using Streptavidin-HRP (Sigma Cat. No. S5512) and TMB singlesolution substrate (Invitrogen Cat. No. 00-2023). The reaction wasstopped with 3M HCl, absorbance read at 450 nm on a Dynex TechnologiesMRX TC II plate reader and the binding curves plotted. IC50 values foreach antibody were calculated and these were normalized to the IC50 ofthe chimera which was included on each respective ELISA plate.

The IC50s obtained show a number of the Composite Human Anti-CLEVER-1Antibodies™ has better binding to CLEVER-1 than the chimeric 3-372.Competition data for the lead variants is shown in FIG. 9.

TABLE 5 Binding characterisation of Composite Human anti-CLEVER-1Antibodies ™ Relative V Region IDs IC50 CH/CK 1.0 VH1/VK1 0.84 VH1/VK21.37 VH1/VK3 1.63 VH1/VK4 1.17 VH1/VK5 1.13 VH2/VK1 0.82 VH2/VK2 0.95VH2/VK3 0.7 VH2/VK4 0.79 VH2/VK5 0.52 VH3/VK1 0.76 VH3/VK2 0.51 VH3/VK3— VH3/VK4 0.47 VH3/VK5 0.42 VH4/VK1 1.86 VH4/VK2 0.9 VH4/VK3 — VH4/VK41.2 VH4/VK5 0.46

The relative IC₅₀ was calculated by dividing the value for the testantibody by that of the chimera assayed on the same plate.

Example 11: Antibody Binding In Vitro

Human peripheral blood monocytes from healthy donors were collected andthey were enriched from about 9 ml of peripheral blood byFicoll-gradient centrifugation. After that they are plated in lowattachment 96-well plates in a density of 1.2×106 cell/well in IMDMmedium supplemented with 1% human AB serum. The cells were treated with1 μg/ml or 10 μg/ml of anti-CLEVER-1 antibody 3-372 (DSM ACC2520deposited at DSMZ-Deutsche Sammlung von Mikroorganismen and ZellkulturenGmbH on Aug. 21, 2001) or VH3/VK5 (a humanized anti-CLEVER-1 antibodyaccording to the present invention recognizing said specific CLEVER-1epitope) for 48 hours. HLA-DR expression was determined from CD14positive cells after 48 hours by using LSR Fortessa flow cytometry. Deadcells were eliminated from the analysis based on the positive signal for7-AAD cell viability dye.

Human IgGs was used as reference.

FIG. 10A shows results of the determination HLA-DR expression from CD14positive cells. HLA-DR expression on CD14 positive cells increased withtreatment of humanized anti-CLEVER-1 antibody VH3/VK5 compared toreference of human IgGs.

No difference in cell viability between treatments was observed. Thus,it can be concluded that the CLEVER-1 targeting antibodies do not affectmonocyte survival.

Example 12: Measurement of TNF-α

Human peripheral blood monocytes from healthy donors were collected andenriched as described in Example 11. Monocytes from 3 ml of erythrocytelysis buffer treated blood were let to adhere overnight on 6-wellplates, washed once with PBS and cultured for 3 days with 10 μg/ml ofanti-CLEVER-1 antibody 3-372 or AK-1.

Soluble TNF-alpha was measured from culture medium using a commercialTNF-alpha ELISA kit (Invitrogen). The results of the measurement areshowed in FIG. 10B. The increased TNF-alpha secretion has noticed bysamples treated with anti-CLEVER-1 antibody compared to untreatedsamples or the control treated samples with AK-1.

Example 13: Mouse Syngeneic Cancer Models

Established E0771 mouse mammary carcinomas were treated with 5 mg/kg ofanti-CLEVER-1 (mStab1) or isotype control every 3-4 days until thetumours reached a size of 1 mm³. The effect of anti-CLEVER-1 treatmenton the recruitment and phenotype of TAMs, different monocyte subsets andtumour-infiltrating leukocytes was assessed using flow cytometry.

FIG. 11A shows TAM re-polarization in syngeneic E0771 mammary carcinomasafter administration of an antibody binding to CLEVER-1. TAMre-polarization is measured by increased macrophage populationsexpressing MHCII (in human HLA-DR) by flow cytometry. Each dotrepresents the percentage of MHCII^(high) CD11b⁺F4/80⁺ TAMs in onemouse. Tumours treated with anti-CLEVER-1 showed a similar level of TAMs(CD11b+F4/80+) compared to the control treated tumours. However, the TAMpopulation in anti-CLEVER-1 tumours consisted of more pro-inflammatorymacrophages (Ly6CloMHCIIhi) with lower expression of the type II marker,CD206.

The anti-CLEVER-1 treated TAMs secreted significantly more TNF-alphacompared to IgG treated TAMs, as shown in FIG. 11B. Each dot representsTAMs isolated from one mouse. Consistent with this, also a decrease inFoxP3+ tumour-infiltrating leukocytes was observed.

The results indicate that CLEVER-1 is a potential target formacrophage-directed immunotherapy.

Example 14

As in example 1 has denoted, the antibodies 9-11 and 3-372 binds todistinct epitopes in human CLEVER-1 and now it has studied the effectsof this difference on signaling in human peripheral blood monocytes.FIG. 12 illustrates that CLEVER-1 ligation with 9-11 and 3-372antibodies promotes opposing effects on mTOR (mechanistic target ofrapamycin) and c-Jun signaling in human peripheral blood monocytes.

FIG. 12A shows flow cytometry analysis of 9-11 and 3-372 binding on CD14positive human monocytes (n=2 donors, D1 and D2).

FIG. 12B shows results, when Human Phospho-Kinase Array (R&D) was usedto measure activation of phospho proteins on CD14 positive cells(enriched by negative selection) after a 10 minute treatment with 20μg/mL of 9-11 and 3-372. The phospho signals were normalized to relevantisotype control treated cells, ratIgG2a for 9-11 and mouse IgG1 for3-372. As shown in FIG. 12B, antibodies 9-11 and 3-372 promote opposingeffects on mTOR and c-Jun signaling in human peripheral blood monocytes.It is known that the mTOR pathway regulates macrophage polarization andimmunosuppressive macrophage phenotype depends on c-Jun phosphorylation,wherein the results indicate that 3-372 antibody activates macrophagesto switch their phenotype from M2 macrophages into M1 macrophages.

OTHER PREFERRED EMBODIMENTS

It will be appreciated that the agent capable of binding to humanCLEVER-1, such as an antibody, the single chain Fv or Fab fragment(s),peptide(s), macromolecule(s), and humanized antibody or humanized singlechain Fv or Fab fragment(s) and pharmaceutical compositions of thepresent invention can be incorporated in the form of a variety ofembodiments, only a few of which are disclosed herein. It will beapparent for the expert skilled in the field that other embodimentsexist and do not depart from the spirit of the invention. Thus, thedescribed embodiments are illustrative and should not be construed asrestrictive.

The invention claimed is:
 1. A humanized antibody or single chain Fv or Fab fragment capable of binding to an epitope of human CLEVER-1, wherein said antibody or single chain Fv or Fab fragment comprises a human IgG heavy chain variable region sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO 18 and SEQ ID NO: 20, and a human IgG light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28 and SEQ ID NO: 30 with the following sequences of complementarity determining regions (CDRs) i) of the heavy chain CDR 1: (SEQ ID NO: 7) TSGMGIG, CDR 2: (SEQ ID NO: 8) HIWWDDDKRYNPALKS, and CDR 3: (SEQ ID NO: 9) HYGYDPYYAMDY; and ii) of the light chain CDR 1: (SEQ ID NO: 10) TASSSVSSSYLH, CDR 2: (SEQ ID NO: 11) RTSNLAS, and CDR 3: (SEQ ID NO: 12) HQYHRSPPT.


2. The humanized antibody or single chain Fv or Fab fragment according to claim 1 capable of binding to human CLEVER-1, wherein the antibody or single chain Fv or Fab fragment binds to one or more sequences selected from the group consisting of (SEQ ID NO: 3) ATQTGRVFLQ, (SEQ ID NO: 4) DSLRDGRLIYLF, (SEQ ID NO: 5) SKGRILTMANQVL, and (SEQ ID NO: 6) LCVYQKPGQAFCTCR.


3. The humanized antibody or single chain Fv or Fab fragment according to claim 1 capable of binding to human CLEVER-1, wherein the human IgG heavy chain variable region sequence comprises SEQ ID NO: 16, SEQ ID NO 18 or SEQ ID NO:
 20. 4. The humanized antibody or single chain Fv or Fab fragment according to claim 1 capable of binding to human CLEVER-1, wherein the human IgG light chain variable region sequence comprises SEQ ID NO: 28 or SEQ ID NO:
 30. 5. The humanized antibody or single chain Fv or Fab fragment capable of binding to human CLEVER-1 according to claim 1, wherein the combination of the human IgG heavy and light chain variable regions is selected from the group consisting of the following combinations: SEQ ID NO: 14 and SEQ ID NO: 22, SEQ ID NO: 16 and SEQ ID NO: 22, SEQ ID NO: 16 and SEQ ID NO: 24, SEQ ID NO: 16 and SEQ ID NO: 26, SEQ ID NO: 16 and SEQ ID NO: 28, SEQ ID NO: 16 and SEQ ID NO: 30, SEQ ID NO: 18 and SEQ ID NO: 22, SEQ ID NO: 18 and SEQ ID NO: 24, SEQ ID NO: 18 and SEQ ID NO: 28, SEQ ID NO: 18 and SEQ ID NO: 30, SEQ ID NO: 20 and SEQ ID NO: 24, and SEQ ID NO: 20 and SEQ ID NO: 30 wherein said antibody, single chain Fv or Fab fragment is capable of binding to human CLEVER-1 with a relative IC50<1.0 in comparison to the IC50 of monoclonal antibody 3-372 (DSM ACC2520 deposited at DSMZ-Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH on Aug. 21, 2001).
 6. An adjuvant for a vaccine comprising an agent capable of binding to human CLEVER-1 according to claim 1, wherein said adjuvant is present in an amount sufficient to remove immune suppression against the vaccine antigens by modulating M2 macrophages into M1 macrophages.
 7. A pharmaceutical composition comprising an agent capable of binding to human CLEVER-1 according to claim 1 in combination with an appropriate excipient.
 8. The humanized antibody or single chain Fv or Fab fragment according to claim 1, wherein the antibody or single chain Fv or Fab fragment binds to the epitope sequences: PFTVLVPSVSSFSSR (SEQ ID NO: 1), and QEITVTFNQFTK (SEQ ID NO: 2) on human CLEVER-1.
 9. The humanized antibody or single chain Fv or Fab fragment according to claim 1, wherein the human IgG heavy chain variable region sequence comprises SEQ ID NO: 18 and the human IgG light chain variable region sequence comprises SEQ ID NO:
 30. 10. The humanized antibody or the single chain FV or Fab fragment according to claim 1, wherein said antibody or single chain FV or Fab fragment comprises constant regions of human IgG4 heavy chain and kappa light chain.
 11. The humanized antibody or the single chain FV or Fab fragment according to claim 1, wherein said antibody or single chain FV or Fab fragment comprises constant regions of human IgG4 heavy chain and kappa light chain with the mutations L248E and/or S241P.
 12. The humanized antibody or the single chain Fv or Fab fragment according to claim 1 capable of binding to human CLEVER-1 with a relative IC50<1.0 in comparison to the IC50 of monoclonal antibody 3-372 (DSM ACC2520 deposited at DSMZ-Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH on Aug. 21, 2001).
 13. The humanized antibody or the single chain Fv or Fab fragment according to claim 12 capable of binding to human CLEVER-1 with a relative IC50<0.8, <0.6 or <0.5 in comparison to the IC50 of monoclonal antibody 3-372.
 14. The humanized antibody or the single chain Fv or Fab fragment according to claim 13 capable of binding to human CLEVER-1 with a relative IC50<0.5 in comparison to the IC50 of monoclonal antibody 3-372. 